SA06270332B1 - hFSF AQUEOUS FORMULATION - Google Patents

Abstract

hFSF AQUEOUS FORMULATION Abstract An aqueous formulation of human follicle stimulating hormone (hFSH) is formulated to maintain hFSH potency for a prolonged or extended period of time. The composition of the present invention is an aqueous formulation comprising a therapeutically effective amount of hFSH fixed in a phosphate buffer containing glycine, methionine and a non-ionic surfactant, preferably polysorbate 20, which is capable of Maintaining effective hFSH for an extended period of time.

Description

11317 hFSF AQUEOUS FORMULATION FULL DESCRIPTION BACKGROUND: The present invention relates to an aqueous formulation of human follicle stimulating hormone (FSH) Ja gail which is stabilized to maintain the potency of hFSH for a prolonged period of time specifically; The present invention relates to an aqueous 117511© formation containing a therapeutically effective amount of 0511 stabilized in a buffer solution of phosphate containing a non-ionic surfactant glycine and methionine. General description of the invention:- 0 WFSH is a reproductive hormone that plays an essential role in FISH functions. It is associated with luteinizing hormone (LH) and human chorionic gonadotropin (026). hFSH «Lily, is a heterodimeric bimolecular glycoprotein formed by non-covalent conjugation of alpha and beta subunits, the alpha subunit consists of 7 amino acid groups while the beta subunit consists of 111 amino acid groups amino acid residues Ye 111 each retaining two glycosylation sites associated with Asn-linked glycosylation sites (Human Reproduction Update 1998, Vol 4, No. 6 pp 862-881) and to 1988 was the primary source L-01511 is a urine-derived FSH isolated from the urine of adult females carrying a child. Another purified form of FSH derived from highly purified urine was introduced in 1998 and finally recombinant FSH was developed and has been used extensively since 1959. FSH wins from the pituitary gland. FSH stimulates the growth and maturation of follicles (ov) ye It is considered an important hormone responsible for ovulation in females FSH Ovary in females. Thus;

Ovum It is used to treat infertility in sterile females who do not ovulate and for artificial insemination. Agpiration/Ovum Pickup (OPU) Generally; Proteins have a very short half-life; And subject to change in ¢ dissociation of dimers LAA dissociation of monomers Jie ee monomers aggregate and adsorption on the surfaces of vessels; After exposure to many factors such as unfavorable temperatures, physical/ mechanical pressure, Jad) to stimulate the activity of proteins; water-air confrontation; Pressure, organic solvents and microbial contamination. As a result; Proteins that change in nature lose their internal physicochemical properties and physiologically active effects. That the proteins change in yaar and the change in nature is not reversible and therefore; 1 their nature may hardly perform their natural properties on the initial state. In particular; Proteins that have a mixed bimolecular structure consisting of two different subunits and are administered in small quantities suffer from fewer than several hundred problems associated with relatively high losses of FSH proteins such as pe μg each including protein losses As a result of the dissociation of dimers in aqueous solutions and the adsorption of proteins on the inner surface of the vessels. The disassembled proteins lose their physiological potency; Monomers are shown

disassembled AE for assembly. And also; The adsorbent proteins are on the inner surface of the vessels to be exposed to aggregation through the process of denaturation. And when it is abused in the human body; Altered proteins in nature may be the cause of the formation of antibodies against naturally occurring proteins. Therefore. The target proteins are supposed to be transactivated in the x state. stable enough. And to this end; A large part of the research has been done on developing a method to prevent denaturation of proteins in solution. Some protein pharmaceuticals have overcome stability problems by lyophilization (freeze drying). as an example; Published US Patent No. 07970091 A lyophilized composition comprising an acid stabilized (ACG) and FSH “TSH” LH (eg gonadotropin on gonadotropin Yo

Yoav

a polycarboxylic acid or a salt thereof; Citric acid and a disaccharide other than ide sucrose are preferred. And published US Patent No. 07590740 a lyophilized composition containing FSH 111 or hCG stabilized by a sucrose-glycine conjugate P8176108. However"; Lyophilized products are unsuitable in that they must be dissolved in Water for Injection (WED) 0 to reconstitute before use. Also, the lyophilized production process includes a freeze-drying step; Thus, it suffers from the need for a major investment such as the use of a high-grade freeze dryer. As an alternative measure to overcome these shortcomings; Protein stability may be improved by adding a protein stabilizer in solution. As examples of useful protein stabilizers; There are 0 known surfactants; Serum albumin Polysaccharides 01755 Amino acids Camino acids Salts and the like. (John Geigert, J.

Parenteral Sci.

Tech., 43, No. 5, 220-224, 1989; David Wong, Pharm.

Tech., October, 34-48, 1997; and Wei Wang, Int.

J.

Pharm., 185, 129- 1 188, 1999 However; The most appropriate stabilizer should be selected and used taking into account the physicochemical properties of the individual proteins; The combined use of different protein stabilizers may result in adverse side effects that conflict with the expected effects. As a result of the competitive effect and the antagonistic interaction between the individual stabilizers. Also, the fixation of the macroproteins in the solution requires good concentration and many efforts; As individual protein stabilizers have a specific YL concentration range acceptable for stabilizing the corresponding proteins (Wei Wang, Int.

J.

Pharm. 185, 129-188, 1999) and at present; Published_US Patent GE) 9078© _formulation of a stable liquid oe liquid formulation of gonadotropins which is prepared by dissolving gonadotropins (eg FSH TSH LH and ACG in a solution consisting of Yo a proven amount of ae polycarboxylic acid or ae gle Yovt

A salt thereof 48 stabilized Su thioether LJ) non-reducing sugar and non-ionic surfactant and according to this field; Sodium citrate is described as the preferred form of polycarboxylic acid or a salt thereof. The composition contains 961.497 0-sodium citrate, and the citric acid solution shows 961 aqueous, strong acidity having VY = pH (Handbook of pharmaceutical excipients 2™ edition, pl24) In order to suit the patient; Those skilled in the field forbade the use of citric acid or a salt thereof. The field mentioned in US Patent No. 9797© suffers from the problem of incompatibility with papal) as a result of pain at the site of administration of the drug resulting from the introduction of sodium citrate 761.57 in the pharmaceutical formulation. on hCG fixed with a phosphate buffer (V = pH) containing a non-reducing polyalcohol or sugar; Preferably Mannitol, Jie Stabilizer. This range of use is based on liquid hCG formulations which are used at a high dose of more than 00001 IU each time; The test has not been conducted on Ally liquid FSH formulations used in a small amount of Ve to You IU as a daily dose. LS, may be noted from the previous fields; There is an urgent need to develop a new liquid formulation 0 that is capable of maintaining the potency of 08511 for an extended period of time. Detailed description: - so; The present invention has been made in light of the foregoing problems; The present invention aims to prepare an aqueous formulation of human follicle stimulating hormone (hFSH) stabilized to maintain hFSH activity for a prolonged or extended period of time. Yo).

-- and here Lad follows; The present invention is described in greater detail. According to the images of the present invention; The foregoing and other objectives may be accomplished by preparing an aqueous formulation (Sle) of human follicle stimulating hormone (hFSH) containing a therapeutically effective amount of hFSH and glycine methionine. Gallll non-ionic surfactant and a phosphate buffer solution as stabilizers.i.e., according to the composition of the present invention, 11511 is stabilized in a phosphate buffer containing glycine©817©10, methionine and a non-ionic surfactant. an enon-ionic surfactant such that the potency of 07511 is maintained for an extended period of time.Yo and as a source of chFSH the aqueous composition of the present invention may use 17511 naturally occurring in the body; or genetic recombinant FSH (FSH) which is formed; isolated and purified from animal cells using genetic recombination technology. Thereby the 17511 that may be used in the present invention includes all species from 10911 comprising both natural and synthetic species. The aqueous formation in the present invention includes glycine as a constituent In the stabilizer, the glycine in solution helps to collect more water molecules around FSH, thereby also stabilizing the hydrophilic extrinsic amino acids among the many amino acids that make up 117511 and as a result stabilize hFSH (Wang, Int.

J.

Pharm. 185 (1999) 129-188) 0 and the glycine content is in the range 0000 to 90680 (w/v); preferably 11 to 9601 (w/v); More preferably 1 to 96 (w/v). The composition of the present invention includes methionine as another component in the stabilizer. Methionine stabilizes hFSH by preventing oxidation of 117511 in aqueous solution (Wang, Int.

J.

Pharm. 185 (1999) 129-188 (Yo).

The methionine content of 8100106 is not in the range 0.000 to BY (wt/pax preferably 1.101 to 961 (wt/v); ve preferably to 960 (w/v). The composition of the present invention also contains a non-ionic surfactant to prevent the adsorption of hFSH onto the surface of the vessel by stabilizing it.Therefore, the 0_surfactant reduces the tension in the protein solution, thus preventing the adsorption or aggregation of proteins on the hydrophobic surface. Preferred examples of a non-ionic pall surfactant that may be used in the present invention may include a non-ionic surfactant (i surfactant on polysorbate) and a non-ionic surfactant. Polysorbate-based non-ionic surfactants These non-ionic surfactants 0 may be used alone or in combination thereof Particularly preferred is a polysorbate-based non-ionic surfactant Specific examples of a polysorbate-based non-ionic surfactant may include Polysorbate 20 71 cpolysorbate ofr polysorbate Te and polysorbate Av, especially polysorbate 20 00. Polysorbate 20 00 is preferred. Polysorbate 20 01 has a relatively less dangerous VO concentration. . Therefore; Polysorbate 20 01 does not reduce or inhibit the adsorption of proteins to the surface even at low concentrations; But it also inhibits the chemical degradation of proteins. The use of non-ionic surfactants of higher concentration in aqueous formation is not appropriate. This is because the use of non-ionic surfactants at a high concentration leads to overlapping effects; This makes it difficult to accurately assess the stability of 0 .- proteins; When the determination of concentration or stability assessment of proteins is done using an analysis method such as spectroscopy - UV, i.e. Isoelectrical Focusing, therefore; The aqueous formation of the present invention contains a non-ionic surfactant at a low concentration of less than 760.1 (mN/vol) and preferably 1.01 to 960007 (w/v).

A= The aqueous formation in the present invention contains a buffer solution of phosphate from 1 mmol to 1 mmol. Preferably, the exponent should be 01 mmol, and more preferably 0% to 50 mmol

Veo is preferred © to the pH of the buffer solution in the range + to the surfactant methionine “glycine” and in addition to the non-ionic °glycine and phosphate buffer described previously; The composition of the present invention may optionally include other components or materials known in the field where they are not harmful to the effects desired in the present invention. For example; The composition may optionally also include benzyl alcohol; kristol; alkyl paraben; and their likes; Which may be commonly used as preservatives for pharmaceutical protein materials. 0 The composition may also include a C-factor. Examples alia and where as the limiting factor of the CFA may include water-soluble inorganic salts such as ssorbitol chloride, mannitol, Jie sodium, and calcium chloride; And sugar alcohols Glucose trehalose Trehalose “maltose Mannose 130006 Maltose dactose Lactose and sucrose ©500:05. These raffinose glycerol 1766:01 p cglucose Vo substances may be used alone or in any combination thereof. The present invention will be described in more detail with reference to the following examples. These examples are prepared to illustrate the present invention only and should not be interpreted as defining the scope and scope of the present invention. Sorbates “glycine to which glycine ml mol was added to which 17511 was then added to 01 to a polysorbate phosphate solution 0 of aqueous formulation (Sle IU / ml), thus preparing the formation of You concentration ml [package of the solution thus prepared into glass bottles ¥ 1 ml WFSH Yo

Yov)

—q- in order. Formula Yo, which was then sealed and isolated at 06, is described below. 1 Structural composition of aqueous formations prepared in the special examples in Table 1. Preparation of aqueous formations of 117511 free of additives. 1: Comparative example ml mol. 01 international units / ml to phosphate solution 050, then add 117511 concentration of a packet of the solution thus prepared into glass containers “ml” which were [da 1] and then sealed 0 were divided and stored at M and 020, respectively

EEE

(Iu/ | glycine | methionine 00 buffered phosphate (Ja polysorbate oe | ovo [ao | os | th [om | vo]) Comparative example 150 10mM | 0 : 1 Unit: mg/mL * - Hydro: hFSH formations stability test: 1 Experimental example at 1 m for a period of 1 to © and comparative example 1 after storing the aqueous formations in the examples; and + months the extraction or oY for yellow OVO deg 1 months yellow a and 0 “SEC-HPLC by hFSH retrieval 9602811 dissociation of 1602511 into monomers and purity at corresponding time points; on Rp-HPLC by %Hfsh alpha of due ill and unit oxidation arrangement. The results obtained are given in the tables “to © below. 1 table sq | om | am | an | om welcome 1

-1.- « | bridging | © | da ve | ow a ha »a »a »go ow lw | Ha 2 (wi

*Based on zero time

im * * a month

schedule? om an] em for “sw] boron] uf ed wa] a]

m * month

° Yovi

-11- table; vee 4 | m | p | d | And

Desa© | E | we [wo | ma month :00 * to “extract or 1 Example configurations showed oF Note from table Ls, sed 7 and ©?has been for more than 9645 under stored configurations when FSH retrieved Retrieving 96 subscripts (~7670) of 101511 under 1 while the comparative example configuration © to ?1 oF example configurations showed the same conditions. In addition; As can be seen from the table + for a period of YO 17511 less than 965 dissociation into monomers under the storage of formations at "m" and the dissociation of 1601511 significantly plus (more than 1 month) while the comparative example showed formation into monomers under the same Conditions. Also, as noted in Table 4; (60 to 9) showed a purity of 17511 more than 9690 under storage of configurations at “m and 1.” Example configurations 0 showed a purity of 11511 low (reduced to 1 while the example configuration showed The comparator “ped 1 5m for a period under the same conditions. From these results, it may be noted that the aqueous composition of (% A + range of yo) and methionine polysorbate includes glycine p17010p8; methionine 1 according to the invention The current enjoys protein loss and denaturation as a result of the adsorption of polysorbate 20 protein on the inner surface of the vessel and prevents protein dissociation into constituent monomers; Ve stabilizes for a prolonged or extended period of time. 3 N1

17 0 Table Shat | sa] at asl wel aa) ven | wl ww] wal Mq of a month: « * To? Range of oxidation 1 As can be seen from Table 0 showed configurations of examples while ¢22Y0 alpha subunit is about 968 to > months when storage of the formations at “m and comparative example 1 showed i.e. formation free of additives; percentage High % of alpha oxidation (about 9615); when storing the formation at 75 °C for a period of 7 months, and of these de il unit (methionine, cglycine, glycine, lel) that the aqueous formations to which Lady results were added; According to the present invention are aqueous formations of polysorbate 20 0 and methionine polysorbate stable [051] that prevent oxidation that may occur after long-term storage. polysorbate methionine cglycine glycine (Y= PH) mmol 01 benzyl alcohol; sodium chloride and lactose were added to phosphate solution 20 0511 was then added to a concentration of 877 IU/mL; Thus, the formation of ally was prepared, and 0 + ml / syringe of the prepared hFSH solution from aqueous formulation sh, which was stored after (Becton-Dickinson) 1 ml, was filled by Ho in glass syringes. That's at ©7 Done. The structural formula of the aqueous formations thus prepared is shown in the following table. lad 1

Yovi yy Comparative examples ¥ to ;: preparation of aqueous formations of highly concentrated 17511. benzyl alcohol to polysorbate 20 01 polysorbate cglycine to which glycine was added optionally sodium chloride and lactose were added (Y = pH) 01 ml mol of a phosphate solution of aqueous formulation without the addition of methionine 00:08 to 161”, thus preparing a manifold formation of 1 ml / syringe from the solution thus prepared into 1.5 glass syringes and FSH © was filled Which was then stored at 220 ml (Becton-Dickinson) and filled by the following. glycine methionine polysorbate jui (international/ml) 20 ewe Jw | | Aha Ke [wo a seq 00s | A.|« Ah cop tah le tah | A A « L L aps © | 0 | [a Tw || a Unit: mg/mL * high concentration experimental hFSH: stability test of aqueous formations from Jha) and comparative examples * to 0 after storage of aqueous formations from examples; and ¢SEC was done -HPLC by %hFSH and + months; oY purity was measured for a zero period.

Goll points at RP-HPLC by hFSH measuring oxidation of the corresponding alpha subunit 96 in the corresponding order. The results thus obtained are given in the following. V Table 1 yim 7 Table (%) oxidized form of alpha subunit purity * (%) aw] aml “| 2m] am] on] amon | os] mel wea] me 1 month m* Example compositions showed; and ©; which included oF notes from Table LS, constituents polysorbate 20 01 polysorbate methionine methionine glycine glycine for stabilizer; purity 07811 more than 9640 up to storage + months at room temperature (75C); © and oxidation of the alpha subunit less than 0096; as measured by SEC-HPLC as measured by

FSH and thus shows that these formations are formations stabilizing the hormone (RP-HPLC) by using FSH and also; the addition of benzyl alcohol as a preservative has no noticeable effects on the stability of more than 96950; but the oxidation of the le unit showed The configurations of the comparative examples?to a remarkable degree of purity, so it is clear that these configurations 9600 sub-alpha 96 were high, exceeding 0, not stable. Industrial use: - in aqueous formulation, as noted from the previous description An aqueous formulation of hFSH of the present invention may consistently maintain the potency of human follicle-stimulating hormone for a prolonged or extended period of time. Additions oY Posted for illustrative purposes;

-M1- Numerous substitutions are considered possible; Without deviating from the scope and extent of the invention as published in the accompanying items.

Claims (1)

PROTECTIONS 1-1 “aqueous formulation” (hFSH) human follicle stimulating hormone Y contains a therapeutically effective amount of chFSH 7 and cglycine methionine reducing For non-jonic surfactant and buffer solution of phosphate as stabilizers; © wherein glycine includes an amount from 100 to 9000 (w/v); methionine 11 includes an amount of 0.010 to Jens) BY volume); The 7 non-ionic surfactant includes an amount from 0.0011 to 960.07 (w/v); And A phosphate buffer solution has a salt concentration of 1 mmol to 50 mmol and 14 0.5 mol to 1 Yo ?-formulation according to the element 1 in which hFSH is naturally occurring 01811 or FSH ¥ recombinant gene (FSH) 1 a formalization according to item 3 for which a non-ionic ¥ surfactant is selected from the group consisting of a surfactant non-ionic surfactant, 7 based on polysorbate 0017507081-08560 » non- {onic surfactant ~~ ¢ based on poloxamer-based and any combination thereof. 1 ¢—formulation according to element oF in which the non-ionic surfactant gal is -polysorbate 20 01 1 #-formulation according to element 1 in which the composition also includes a preservative 1 V or an isotonic agent. Tov) 1 = the formation according to the element © in which the preservative is ¥ cbenzyl alcohol ccresol alkyl paraben or a mixture of them thereof |.” and the frequent factor isotonic agent is a water-soluble inorganic salt; s water-soluble inorganic salt | or .sugar alcohol